Treatment of protein with acids and product resulting therefrom



Patented May 20, 1947 TREATMENT OF PROTEIN WITH ACIDS AND PRODUCTRESULTING THEREFROM John Robert Coflman, St. Paul, Minn, and Harry F.Lewis, Appleton, Wis., assignors, by mesne assignments, to GeneralMills, Inc., a corporation of Delaware No Drawing. Application August23, 1945, I

Serial No. 612,307

Claims.

The present invention relates to modified pro teins and to a process ofeffecting such modification and more particularly to the modification.of proteins for use in plastics having low water absorption properties.

The present application is a continuation in part of our co-pendingapplication, Serial No. 448,292, filed June 24, 1942.

Numerous attempts have been made to modify proteins to make themsatisfactory for use as plastics, in view of the low cost of suchproteins and in view of their ready availability. In general, most ofthese protein plastics have proved unsuccessful commercially except invery limited circumstances. They absorb excessive quantities of waterand as a result lose their a shape. In some instances this excessiveabsorption of water also results in such a loss of strength that theproducts disintegrate very readily. These disadvantages are emphasizedin certain uses of plastics such, for example, as machine parts in whichclose tolerances are required. The

protein plastics of the prior art lose their shape to such an extentwhen they absorb water that they are entirely unsuitable for such uses.

It has now been discovered that proteins may be treated according to thepresent invention to produce a product of materially reduced waterabsorption. In general, the water absorption of protein after treatmentaccording to the present invention is approximatelyhalf of what it wasprior to treatment.

It is, therefore, the principal object of the present invention toprovide a modified protein having low water absorption properties. It isa further'object of the present invention to provide a method ofproducing such modified protein.

These and other objects of the invention will be apparent from thfollowing description thereof with particular reference to specificexamples which are given for the purpose of illustration and which arenot to be construed as limiting the invention.

The invention is applicable to proteins in general, such as casein,soybean protein, zein, Wheat gluten, and the like, whether isolated frommaterials with which they naturally occur or in ad-' I mixture withother materials. For purposes of the present invention the treatmentwill be described with particular reference to wheat gluten, it beingunderstood that similar treatment is applicable to other proteins.

The invention involves the'treatment of proteins with relativelyconcentrated solutions of highly ionized mineral acids such ashydrochloric acid and sulfuric acid, in combination with nitrous acid.This treatment which is referred to herein as nitrous acid treatment,completely hydrolyzes all ammonium salt groups and all primary amidegroups, and at the same time converts primary amine groupsto hydroxylgroups.

Analytical data seem to point to the fact that this treatment affectsthe protein chemically in the manner described ,above. Thus, the basebinding capacity of the treated protein is materially higher than thatof the untreated protein, indicating hydrolysis of ammonium and primaryamide groups to carboxyl groups. Formol titration of the nitrous acidtreated proteins shows a decrease in primary amine groups indicating theconversion of primary amine groups to hydroxyl groups. The conversion ofprimary amine groups to hydroxyl groups and the conversion of primaryamide groups to carboxyl groups is further indicated by the evolution oflarge quantities of nitrogen, which is characteristic of thesereactions.

The treatments herein described appear to be efiected without anyappreciable hydrolysis of the peptide bonds. Formol titration, whichmeasures primary amine groups, when applied to the starting material andto the material treated with mineral acid in the absence of nitrous acidshows no change in the content of the primary amine groups. Sincehydrolysis of peptide bonds would result in an increase in primary aminegroups it was concluded that no hydrolysis of peptide bonds hadoccurred. Since nitrous acid results in a destruction of primary aminegroups it is drochloric or sulfuric in which the hydrogen ionconcentration is not appreciably lower in con- ;centration than IN. Thetreating acid is preferably obtained by suspending the protein in anacqueous solution of a soluble nitrite such as sodium nitrite and thenmaking the suspension acid with, for example, hydrochloric acid insufficient quantity and of sufficiently high concenon t ation that thehydrogen ion concentration in the mixture will not be appreciably lowerthan IN. The suspension of protein in acid is Dreierably allowed tostand at room temperature for an extended period of time, as forexample,20 hours. Thereafter, the material is heated to an elevated temperaturepreferably within the range of 50-80 C., for example 75 C. and theinsoluble material separated from the acid. This treatment at anelevated temperature is for the purpose or completing the reaction andshould be of short duration, for example, 5-15 minutes. This insolublematerial is granular, and can be readily washed to remove acid and otherimpurities. Part of the protein becomes soluble in the acid and can beseparated therefrom by adjusting the pH to a suitable higher value. Theseparated proteins can be suitably processed for removal of adherentacid and other impurities to yield the desired products.

Example 1.HON gluten 500 grams of fat-free gluten were suspended inthree liters of an aqueous solution containing 104 grams of sodiumnitrite and the suspension was stirred for three hours at roomtemperature. To this suspension there were added in a dropwise manner2'73 cc. of 12N HCl over a period of 1.5 hours. The acidified solutionwas allowed to stand over night, heated to 75 C. and filtered. It waswashed by three one-liter portions of water. The gluten was subjected toa second washing with two more liter of water and filtered. The washedgluten was stirred with two liters of water, the suspension adjusted toa pH of 3.0, and filtered. The gluten was dehydrated with two liters ofacetone and dried at 50 C. The dried material when suspended in watergave slight chloride ion tests. It was therefore rewashed with wateruntil the washings were free of chloride ion. The-dried product weighed315 grams. This recovery amounts to 66.4% of the original weight of thegluten. The base binding capacity of the original gluten was 31 whilethat of the nitrous acid gluten was 111. These and other base bindingcapacities are expressed as equiva1ents 10-- per gram. An additionalquantity of nitrous acid gluten may be recovered from the acid filtrateand the various wash waters by pH adjustment, for example to pH valuesof 2.0,'3.5, 4.0, and 5.5. This material requires more extensive washingfor removal of water solubles, but the final product appears to difierprincipally from the acid insoluble material in its base bindingcapacity, which was found to be 166.

Example 2.-HONO-casein, zein, and soya protein products 250 grams ofeach of the proteins casein, zein, and soya protein were suspended in1.5 liters of 0.5N sodium nitrite solution and stirred for three hoursat room temperature. To this suspension there was then added, in adropwise manner, 136 cc. of 12N hydrochloric acid while the proteinsuspension was well stirred. The resulting mixture was allowed to standeighteen hours at room temperature. It was then heated to 75 C.,filtered, and washed well with water until the washings gave only aslightly positive test for chloride ions. The product was drained welland dried at 70 C. for twenty hours. In the case of casein and soybeanprotein, the manipulation was simple throughout and the product appearedmealy in all stages of the procedure. The zein tended to form a cake,floating on the surface. However, agitation served to break up the cakeinto granular particles. Following the reaction the cake which wasproduced was placed in a Waring Biendor and disintegrated in water sothat the resulting material was like fine sand.

As was pointed out above, concentrations of acid appreciably less than1N do not appear to effect the desired results to any substantial extent. The N acid should be used in excess, usually not less than about 2parts by weight of N acid to 1 part by weight of protein. Any largerratio of acid to protein may be employed. Similarly, more concentratedacid may be employed, but in general concentrations materially in excessof about 3N should be avoided as they tend to solubilize the protein;The temperature of reaction is preferably maintained low for a largepart of the time and is elevated only near the end of the reaction. Thetime period may be shortened somewhat by conducting the reaction attemperatures above room temperature. Temperatures in excess of 40 C.should, however, be avoided during the major portion of the reaction inview of the undesirable effect of the acid on the protein when subjectedto such temperatures for any extended period of time and in view of theundesirable efiect of the elevated temperature on the nitrous aciditself.

It will be apparent from the above examples that the product hereindescribed results in a decided improvement in the characteristics of theprotein, thus making the material more useful 'in instances where waterabsorption is an importaht factor. It will also be apparent that theinvention is not limited to the specific examples given above and thatvarious modifications may be made without departing from the spirit ofthe invention as defined in the appended claims.

We claim as our invention:

1. Process for the modification of protein to reduce its waterabsorptive properties which comprises treating said protein with atreating agent comprising a mixture of nitrous acid and a highly ionizedstrong mineral acid selected from the group consisting of hydrochloricacid and sulfuric acid, the hydrogen ion concentration of the treatingagent being not substantially less than 1N, for an extended period toconvert ammonium groups and primary amide groups to carboxyl groups, andto convert primary amine groups to hydroxyl groups without extensivehydrolysis of the peptide bonds.

2. Process for the modification of wheat gluten to reduce its waterabsorptive properties which comprises treating said wheat gluten with atreating agent comprising a mixture of nitrous acid and hydroxyl groupswithout extensive hydrolysis of the peptide bonds.

3. Process for the modification of rein to reduce its water absorptiveproperties which comprises treating said zein with a treating agentcomprising a mixture 01 nitrous acidand a hi ly ionized strong mineralacid selected from the group consisting oi hydrochloric acid and sul-Iuric acid, the hydrogen ion concentration of the treating agent beingnot substantially less than 1N, tor an extended period to convertammonium groups and primary amide groups to carboxyl groups, and toconvert primary amine groups to hydroxyl groups without extensivehydrolysis of the peptide bonds. r

4. Process tor the modification of soy protein to reduce its waterabsorptive properties which comprisestreating said soy protein with atreat-- ing agent comprising a mixture of nitrous acid and a highlyionized strong mineral acid selected from the group consisting ofhydrochloric acid and sulfuric acid, the hydrogen ion concentration ofthe treating agent being not substantially less than 1N, for an extendedperiod to convert ammonium groups and primary amide groups to carbonylgroups, and to convert primary amine groups to hydroxyl groups withoutextensive hydrolysis oi the peptide bonds.

5. Process for the modification 0! protein to reduce its waterabsorptive properties which comprises treating said protein withatreating agent comprising a mixture of hydrochloric acid and nitrousacid, the hydrogen ion concentration of the treating agent being notsubstantially less than 1N, for an extended period to convert ammoniumgroups and primary amide groups to carboxyl groups, and to convertprimary amine groups to hydroxyl groups without extensive hyrirolysis ofthe peptide bonds.

6. Process for the modification of protein to reduce its waterabsorptive properties which comprises treating said protein with atreating agent comprising a mixture of hydrochloric and nitrous acids,the hydrogen ion concentration of the treating agent being notsubstantially less than 1N for an extended period 0! time at atemperature not substantially in excess or 40' C. and then raising thetemperature or the reaction mixture to the approximate range of 50-80"C. for a short period or time, whereby ammo um groups and primary amidegroups are converted to carboxyl groups and primary amine groups areconverted to hydroxyl groups without substantial hydrolysis of thepeptide bonds. v

7. A modified protein characterized by reduced water absorption, saidprotein having a major proportion of its peptide bonds intact and havingits primary amine groups converted to hydroxyl groups and having itsammonium groups and primary amide groups converted to carboxyl groups.

8. A modified wheat gluten characterized by reduced water absorption,said wheat gluten having a major proportion of its peptide bonds intactand having its primary amine groups converted to hydroxyl groups, andhaving its ammonium groups and primary amide groups converted tocarboxyl groups.

9. A modified zein characterized by reduced water absorption, said zeinhaving a major proportion of its peptide bonds intact and having itsprimary amine groupsconverted to hydroxyl groups, and having itsammonium groups and primary amide groups converted to carboxyl groups.

10. A modified soy protein characterized by' reduced water absorption,said soy protein having a major proportion of. its peptide bonds intactand having its primary amine groups converted to hydroxyl groups, andhaving its ammonium groups and primary amide groups con verted tocarbonyl groups. JOHN ROBERT comm.

HARRY F. LEWIS.

REFERENCES CITED The following. references are of record in the .file ofthis patent:

cm'rnn s'ra'rns PATENTS Number Name Date 2,158,117 Grettie May 16, 1939

